Netrin‐1 upregulates GPX4 and prevents ferroptosis after traumatic brain injury via the UNC5B/Nrf2 signaling pathway

Abstract Aim We aimed to investigate the regulatory role of Netrin‐1 (NTN1) in ferroptosis after traumatic brain injury (TBI) in mice. Methods We assessed the expression pattern of NTN1 by RT–PCR, western blot, and immunofluorescence after establishing the TBI model in mice. After treatment with NTN1 shRNA or recombinant NTN1, we determined the biochemical and morphological changes associated with ferroptosis and netrin‐1‐related pathways. We used Nissl staining to assess lesion volume and Morris water maze and beam‐walking test to evaluate ethological manifestation. Results The mRNA and protein levels of NTN1 were upregulated after TBI. The application of NTN1 shRNA increased the number of FJB positive cells, malondialdehyde (MDA), and reactive oxygen species (ROSs) levels. However, the application of NTN1 recombinant had the opposite effect. Furthermore, knockdown or inhibition of GPX4, Nrf2, and UNC5B counteracted the effects of NTN1 recombinant. Intravenous injection of NTN1 recombinant reduced neuronal loss after CCI and improved motor and cognitive function. Conclusion NTN1 had a neuroprotective effect after TBI and inhibited ferroptosis via activating the UNC5B/Nrf2 pathway. These findings may provide potential therapeutic strategies for TBI.


shRNA treatment and intracerebroventricular injection
In vivo gene knockdown was achieved by Adeno-associated virus 9 (AAV9) based short hairpin RNA (shRNA) and in vitro gene knockdown was achieved by lentivirus-based shRNA. In brief, we constructed the recombinant plasmid by inserting cDNA between the inverted terminal repeats of AAV9 or lentivirus genome. Helper plasmids were cotransfected with recombinant plasmid into 293T cells to package the vector. The vector was harvested at 48 h post-transfection and was purified by virus purification kit (Cell Biolabs, USA). Virus titer was determined using the Rapid Titer Kit (Clontech, USA). 1×10 10 copies of AAV9-Vector were injected into the lateral ventricle 14 d before CCI and lentivirus was added to the media at a MOI of 100 72 h before modeling. The target sequences were presented in Supplementary Table 1. The knockdown efficiency was validated by WB. Intracerebroventricular (ICV) injection was performed as previously described 20 with some modifications. Mice were anesthetized and placed in a stereotaxic apparatus. The stereotactic coordinates were as follows: anteroposterior (AP), −0.4 mm; mediolateral (ML), −1.0 mm; dorsoventral (DV), −3.0 mm from the bregma for injection into the left lateral ventricle. Animals were injected with 10 μl using a syringe with a 0.52 mm needle. Virus was injected over 10 min, and the needle was left in place for 10 min prior to withdrawal.
Two weeks were needed for successful transfection before CCI.

RNA-sequencing
The samples were obtained from the injured cortex 3 days after CCI and processed for RNA sequencing. Specifically, cortex were collected after saline perfusion and washed in PBS. The tissue was placed into pre-chilled RNase-free tubes and frozen at −80°C.
Following sequencing procedures were performed by Majorbio Co. Ltd (Shanghai, China).

Slice preparation
The mice were anesthetized and then transcardially perfused with saline (4°C ) and 4% paraformaldehyde (4°C ) sequentially. The skin and skull were stripped, and the whole brain was harvested, fixing in 4% paraformaldehyde (4°C ) for 6 hours. After that, the brains were dehydrated sequentially in 20% and 30% sucrose solution (4°C ) for 24 hours. Finally, the brain tissues were cut into 20 μm-thick serial coronal sections and stored at -80°C.

Transmission electron microscopy (TEM)
TEM was used to examine the alterations in mitochondrial morphology of the injured neurons. The mice were anesthetized and then transcardially perfused with saline. The brain tissues were quickly removed and placed in the electron microscope fixing solution (Simuwu, China) for 4 hours. After 3 times rinsing with 0.1 M phosphate buffer (PB), fixing in 1% osmium acid/0.1 M PB (PH7.4) for 2 h (20°C), 3 times rinsing with 0.1 M PB, the tissues were sequentially dehydrated with 50%, 70%, 80%, 90%, 95%, 100%, 100% alcohol and 2 times 100% acetone. The tissue samples were then oriented and placed in a 60 °C oven for 48 h. The ultrathin sections (60-80 nm) were cut with an ultramicrotome and stained by 2% uranium acetate saturated alcohol solution and lead citrate for 15 minutes. Sections were dried at room temperature and examined under a transmission electron microscope.

Measurement of malondialdehyde (MDA), reactive oxygen species (ROS), and glutathione (GSH) levels
MDA assay kit (S0131, Beyotime, China) was used to measure the MDA content in mouse brain tissue. The principle of this kit is the color reaction of MDA and thiobarbituric acid to give a red product, having a maximum absorbance at 535nm. The ipsilateral cortex tissues were sufficiently broken and lysed on ice for 30 minutes, and then centrifuged at 12000g for 10 minutes to take the supernatant. BCA kit was used to detect the total protein concentration. According to the kit instructions, the absorbance was measured at 500nm and the MDA content was expressed as nmol/mg prot. GSH assay kit (S0053, Beyotime, China) was used to measure the GSH content. The principle of this kit is that GSH can react with the chromogenic substrate DTNB to produce yellow TNB and GSSG. Briefly, we collected the ipsilateral cortex tissues, measured the absorbance at 412nm and calculated the GSH content by standard curve according to the instructions.
The principle of the kit is that DCFH-DA probe can be oxidized by ROS into the fluorescent substance DCF. The absorbance was measured at 500nm and the ROS content was expressed as U/mg prot.

Measurement of Netrin-1 levels in cerebrospinal fluid (CSF) of mice
The cerebrospinal fluid of mice was taken to determine the content of Netrin-1. The mice were sacrificed by CO2 inhalation and the foramen magnum was exposed. We punctured a hole at the base of the skull and obtained the CSF. Only the clear liquid was collected for further use. The CSF was then harvested by centrifugation. Netrin-1 assay kit (SEB827Mu, Cloud-Clone, China) was used to evaluate the Netrin-1 content. Briefly, Netrin-1 antibody was precoated onto 96-well microplates to prepare a solid-phase carrier and the determination of Netrin-1 concentrations was performed by ELISA test. We measured the OD value at 450nm wavelength and calculated the Netrin-1 content by standard curve according to the instructions.

Western-blot and nuclear-cytoplasm separation
Western-blotting was used to assess specific protein levels. For mice, the ipsilateral cortex tissues were sufficiently broken and lysed in RIPA buffer and PMSF with sonication.
Samples were lysed for 30 min on ice and centrifuged at 12500 g for 30 min at 4°C to collect the supernatant. For the cells, RIPA buffer and PMSF were added to each culture dish after PBS rinsing. Cells were harvested using a scraper 5 min later, collected into a tube, lysed for 30 min on ice and centrifuged at 12000 g for 10 min at 4°C to collect the supernatant.
After collection, BCA assay kit (Beyotime, China) was used to detect the protein concentrations. The extracted proteins were separated by electrophoresis with the 10% SDS-PAGE and transferred into a polyvinylidene difluoride (PVDF) membrane in a wet electron transfer device. The membrane was blocked at room temperature for 1 h with 5% non-fat milk, incubated with primary antibody overnight at 4°C, washed with TBST three times and then incubated with secondary antibody for 1 h at room temperature. Protein signals were visualized using ECL luminescent detection system. Band density was quantified by Image J software and data were normalized to β-Actin. The following primary antibodies were used: monoclonal rabbit anti-Netrin-1 (ab126729, 1:1000, Abcam, UK), monoclonal rabbit anti-GPX4 (ab125066, 1:1000, Abcam, UK), monoclonal rabbit anti- Proteins from different subcellular fractions were prepared by a nucleoplasmic separation kit (P0027, Beyotime, China). Briefly, the cell membrane ruptured due to low osmotic pressure conditions in this method, releasing the cytoplasmic proteins. Nuclei were precipitated by centrifugation and western-blotting was used to evaluate the protein levels.

Quantitative real-time PCR (qRT-PCR)
qRT-PCR was used to quantify the mRNA levels of NTN1, GPX4, and Nrf2. Briefly, the ipsilateral cortex tissues or the SH-SY5Y cells were collected and total RNA was extracted using Trizol reagent (Invitrogen, USA). cDNA was synthesized using 1 μg of total RNA with PrimeScript Reverse Transcriptase (Takara, Japan). The qPCR reaction was performed based on the primers (Sangon, China) and the RR420A kit (Takara, Japan). qPCR was performed using the following thermo-cycling procedure: 95˚C for 5 sec, 60˚C for 30 sec, repeated for 40 cycles. Each sample was detected 3 times and the mean value was obtained. β-Actin was used as an internal control. The primers are presented in Supplementary Table 2.

Dual-luciferase reporter gene assay
Dual-luciferase reporter gene assay was used to determine the GPX4 promoter activity. The GPX4 promoter sequence was amplified from pcDNA3.1-GPX4 plasmid and cloned into pGL3 luciferase reporter vectors (Youbio, China). The SH-SY5Y cells were cotransfected with the constructed pGL3 and pRL-TK vector (Youbio, China) for 24h and lysed according to the instructions of Dual Luciferase Reporter Assay Kit (Vazyme, China).
Absorbance of the opaque 96-well plate was read at a wavelength of 350-700 nm using SpectraMax M2 spectrophotometer (Molecular Devices, USA). Three replicate holes were set for each sample and the pRL-TK vector was used as an internal control. The values were normalized to the Ctrl group.

Immunofluorescence staining
Immunofluorescence staining was used to visualize the protein localization. The brain sections or the petri dish containing the adherent cells were fixed with 4% paraformaldehyde for 15 min at room temperature, washed with TBST 3 times, permeabilized with 0.2% Triton X-100/TBST for 20 min, washed with TBST 3 times again,

Nissl staining
Nissl staining was used to evaluate the damaged area 3 weeks after CCI modeling.
The sections were washed with distilled water twice and stained with Nissl staining solution (C0117, Beyotime, China) for 30 min at room temperature. After washing with distilled water twice, dehydrating with 95% ethanol twice, and transparentizing with xylene twice, the sections were sealed with neutral gum. We evaluated the lesion area by Image J software. Specifically, we added the lesion area of each slice and multiplied it by the slice thickness to estimate the lesion volume.

Morris water maze
Four weeks after CCI modeling, the learning and memory functions were evaluated using the Morris water maze test. The maze was a circular pool with a diameter of 120 cm and a depth of 50 cm. There was a white platform with a diameter of 6 cm and a height of 30 cm on the southwest quadrant of the pool. The pool was filled with water and titanium dioxide was added to the water to make the water white. The platform was immersed 1 cm below the water surface. In each trial, a mouse was positioned in one of the four directions (east, south, west, and north) facing the wall and then released to swim. There were two criteria for terminating the trial: the mouse found the platform and rested on the platform for 10 seconds, or the mouse did not find the platform within 60 seconds. Each mouse was trained for 5 consecutive days, four times per day, with 4-minute interval between trials. On the sixth day, the camera was placed above the maze to record the movement of each mouse, and the track analysis software was used to calculate the results, including swimming distance, latency and swimming speed.

Beam-walking test
Beam-walking test was used to evaluate the motor function of mice 7 days after CCI modeling. The beam was a square wooden stick with a length of 1 m and a width of 14 mm, whose one side was placed in a black square box. In each trial, a mouse was placed on the other side of the beam so that it entered the square box. Each mouse was trained for two consecutive days, three times per day, with 2-hour interval between trials. On the third day, the walking time and the number of slips for the mouse to enter the box through the beam in 60 s were recorded. Mice that failed to enter the box or the walking time exceed 60 s were both recorded as 60 s.   (a-b) Histogram shows the quantitative analysis of Acsl4 and LOX protein content determined by western blot. The data for each group conformed to a normal distribution. P value was determined by ANOVA with LSD post-hoc test. **P < 0.01. There was no difference in body weight between mice in each group. n=5 (CCI group and CCI+shNTN1 group, 1 mouse died respectively), n=6 (sham group and CCI+shScr group). (c-d)

Supplementary
Histogram shows the quantitative analysis of Acsl4 and LOX protein content determined by western blot. The data for each group conformed to a normal distribution. P value was determined by ANOVA with LSD post-hoc test. **P < 0.01. There was no difference in body weight between mice in each group. Sham group: n=6 each. CCI group: n=5 (CCI+15 μg/kg NTN1 group, 1 mouse died), n=6 (CCI+PBS group, CCI+5 μg/kg NTN1 group and CCI+45 μg/kg NTN1 group). (e-g) MDA, ROS and GSH levels in the mice injuried cortex.